Defining the genome content of live plague vaccines by use of whole-genome DNA microarray

Vaccine 22 (2004) 3367–3374

Defining the genome content of live plague vaccines by use of whole-genome DNA microarray Dongsheng Zhou1 , Yanping Han1 , Erhei Dai, Yajun Song, Decui Pei, Junhui Zhai, Zongmin Du, Jin Wang, Zhaobiao Guo, Ruifu Yang∗ Laboratory of Analytical Microbiology, National Center for Biomedical Analysis, Army Center for Microbial Detection and Research, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences (AMMS), Beijing 100071, PR China Received 14 October 2003; received in revised form 26 February 2004; accepted 27 February 2004 Available online 28 March 2004

Abstract Yersinia pestis whole-genome DNA microarrays were developed to perform genomic comparison of a collection of live plague vaccines. By using the genomic DNA to probe the DNA microarrays, we detected dozens of deletions and amplifications of the genomic regions in the 19 vaccine strains analyzed. The revealed genomic differences within the vaccine strains of different origins provide us an unprecedented opportunity to understand the molecular background of the variability of the immunogenic and protective potency of plague live vaccine. The whole-genome DNA microarray also provides an ideal tool to perform the pre-evaluation of a vaccine strain for its high throughput to determine the genomic features essential or unallowable for the live vaccines. © 2004 Elsevier Ltd. All rights reserved. Keywords: Yersinia pestis; Live plague vaccines; DNA microarray

1. Introduction Plague, one of the most devastating diseases of human history, is caused by Yersinia pestis. Endemic areas for this disease widely exist in Asia, Africa and America, where the occasional epizootics of animal plague pose great threats to public health [1]. Plague has been classified as a reemerging disease by the Word Health Organization due to the worldwide increasing incidence of human plague. Cases of bubonic plague can be well controlled by timely antibiotic treatment. However, the pneumonic or septicemic plague is difficult to be treated with antibiotic therapy. The recent isolation of a multiple antibiotic resistant strain of Y. pestis indicates that the longer term potential for the use of antibiotics to treat plague is less certain [2]. The public health threat posed by plague is thought to be only reliably encountered by use of the effective vaccines. Both live attenuated and killed whole cells vaccines have been used in human [3–5]. The sub-unit vaccines based on the F1 and V antigens take promis∗ Corresponding author. Tel.: +86-10-66948595; fax: +86-10-83820748. E-mail address: [email protected] (R. Yang). 1 Zhou D. and Han Y. contributed equally to this work.

0264-410X/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2004.02.035

ing for their efficacy against both bubonic and pneumonic plague when tested recently in animal models of the disease [4,5]. The large-scale genome sequencing effort and the ability to immobilize thousands of DNA fragments on a solid surface, such as coated glass slide, have led to the development of DNA microarray technology. While the DNA microarray-based comparative genome analysis on many strains of several pathogenic species has contributed to our understanding of bacterial diversity, evolution and pathogenesis [6,7]. Behr et al. described the DNA microarray-based genomic comparison of Mycobacterium tuberculosis with closely related Mycobacterium bovis, and with Baccile Calmette-Guérin (BCG) strains that were produced by serial in vitro passages of M. bovis [8]. The microarray procedure was used successfully to analyze the deletions and amplification mutations found in Salmonella mutagenicity assay strains (Ames test) [9]. The Escherichia coli K-12 MC4100, an MG1655 derivant, has provided an important host in early gene expression study, while Peters et al. used a whole-genome array based on the genome sequence of strain MG1655 as a tool to identify the deletions in MC4100 [10]. The DNA microarray technology represents an ideal methodology to compare or define the contents of the closely related genomes.

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Although live attenuated plague vaccines were developed and tested in human nearly one century ago [11,12], there are still some severe side-effects and the questionable efficacy in evoking response in humans. The virulence of vaccine strains differs from species to species with chronic infections occurring in nonhuman primates. Phenotypic variations were observed within the various derivants from the strains with the same origin. The recent determination of the whole-genome sequences of Y. pestis CO92 [13], KIM [14] and 91001 (Song and Yang, unpublished) provides a framework for the genomic analysis of live plague vaccines. In our present work, we developed DNA microarrays representing nearly all open reading frames (ORFs) annotated in the three finished sequences, and used the microarrays to perform full-genome comparative analysis on a collection of strains of live plague vaccines. By using the genomic DNA to probe the whole-genome DNA microarrays, we detected deletions of a genomic region less than a kilobase in size which may contain only one gene. The amplifications of large genomic regions were also revealed by this kind of microarray analysis. The whole-genome DNA microarray provide an important tool to extend the genome content of the finished Y. pestis strains to other ones, herein the live plague vaccines. The revealed genomic differences within the vaccine strains of different origins provide us an unprecedented opportunity to understand the molecular background of the variability of the immunogenic and protective potency of live plague vaccine. The genomic variability in live plague vaccines suggest us the necessity of re-evaluating the efficacy of the vaccines. The whole-genome DNA microarray also provides an ideal tool to perform the pre-characterization of a vaccine strain for its high throughput to determine the genomic features essential or unallowable for the live vaccines.

2. Materials and methods 2.1. Bacterial strains Nineteen live vaccine strains of Y. pestis were used in this study (Table 1). These strains have been historically tested in human immunization or used in plague research. All the vaccines, before used to prepare DNA for this study, have been stored in dry at −70 ◦ C since 1960s. In addition, two natural isolates of Y. pestis, 91001 and 82009, were used as reference strains in microarray analysis. Y. pestis 91001, a human avirulent strain of biovar Mediaevalis, is isolated from a Micortus-related plague endemic area in China. Y. pestis 82009, a fully virulent strain of biovar Orientalis, is isolated from a house mouse-related plague endemic area in China and was used as an alternative of CO92 that is also an Orientalis one (see below). Moreover, thirty six natural isolates of Y. pestis were used in the PCR screening. These thirty six strains, covering all the three biovars of Y. pestis [1], were isolated from ten kinds of plague endemic areas in China, elaborately selected to represent the most abundant natural populations of Y. pestis. All the vaccines are now collected in our laboratory, while the natural isolates in Qinghai Center for Disease Prevention and Control (QCDPC), China. Strains were grown in Luria–Bertani broth and then genomic DNA was extracted from the bacterial culture as described previously [14]. 2.2. Y. pestis whole-genome DNA microarray In the present work, 4005 annotated ORFs (genes) were amplified successfully from Y. pestis 91001 or 82009 by using gene-specific primer pairs. The word “gene” will be used in the following part of this paper in reference to the ORF.

Table 1 Live plague vaccines used in this study Strain

Institution of collection

Features

EV76 EV76 (B.SHU) EV EV (SHU) EV (Yuan) EV76 (Paris) EV40 Tjiwidej (rough) Tjiwidej (smooth) M23 A1122 P175 Sussia M. II Otten 55 105 410041 P2

Academy of Military Medical Sciences (AMMS), China Qinghai Center for Disease Prevention and Control (QCDPC), China AMMS QCDPC QCDPC QCDPC Chinese Medical Culture Collection (CMCC) CMCC AMMS QCDPC QCDPC AMMS AMMS AMMS AMMS AMMS AMMS AMMS AMMS

Pgm− Pgm− Pgm− Pgm− Pgm− Pgm− Pgm− Pgm− Pgm− Pgm− Pgm− Pgm− Pgm− Pgm− Pgm− Pgm− Pgm− Pgm− Pgm−

pCD1− pCD1− pCD1− pPCP1− pCD1− pCD1− pCD1− pCD1−

pPCP1− pPCP1− pPCP1− pPCP1−

D. Zhou et al. / Vaccine 22 (2004) 3367–3374

These 4005 genes included nearly all the CO92 genes and the 91001-unique genes after the exclusion of genes encoding IS protein, integrase, and transposase. The purified PCR products were spotted on the CSS-1000 silylated glass slides (CEL) by using a SpotArray72 Microarray Printing System (Perkin-Elmer Life Sciences) to construct the DNA microarrays. The genomic DNA mixture of 91001 and 82009 with equal quantity was used as Reference DNA. Genomic DNA from each of the live vaccines studied was referred to as Test DNA. Cy3- or Cy5-labeled probes were generated by priming of the Reference or Test DNA with random hexamers and extension with Klenow [8]. The labeled Reference and Test DNA were combined to hybridize together with the

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microarrays by methods of two-fluorescence hybridization [8]. All hybridizations were performed in triplicate. The hybridized slides were scanned by using a GenePix Personal 4100A Microarray Scanner (Axon Instruments). The scanning images were processed and the data were further analyzed by using GenePix Pro 4.1 software (Axon Instruments) combined with Microsoft Excel software. 2.3. Identification of deletions and amplifications A ratio of intensity (Test DNA normalized intensity/Reference DNA normalized intensity) was recorded for each spot and then was converted to log (2.5). The

Table 2 RDs identified in the 19 vaccine strains studied RD

Gene region

Function

Absent in natural isolates tested

Genes selected for PCR validation

RD01 RD02

pCRY (91001) pPCP1(CO92)

Yes Yes

pCRY03 YPPCP1.06

RD03 RD04 RD05 RD06 RD07 RD08

pCD1(CO92) pMT1(CO92) YPMT1.72c–1.78(CO92) YPMT1.81c–1.84(CO92) pMT044–047(91001) pMT086–093(91001)

Yes No No No Yes Yes

YPCD1.30c YPMT1.86c YPMT1.74 YPMT1.84 pMT045 pMT092

RD09 RD10

pMT127–128(91001) YP0966–0986(91001)

Type IV secretion system Plasminogen activator (Pla), pesticin and pesticin immunity protein Type III YOP protein secretion system Replication and partition related proteins; hypothetical proteins Yersinia murine toxin (Ymt) F1 antigen Ribonucleoside-diphosphate reductase C-type natriuretic protein, DNA ligase and hypothetical proteins Hypothetical proteins Two-component regulatory system, multidrug transport system, tetrahydromethanopterin reductase; NADH:flavin oxidoreductases

Yes Yes

pMT127 YP0975

RD11-1

YPO0568–0618(CO92)

A genomic island containing adhesion, autotransporter, protein kinase, resistance proteins and secreted proteins

Partially absent

YPO0573, YPO0586, YPO0620, YPO0623, YPO0624, YPO0636 and YPO0641

RD11-2 RD11-3 RD11-4

YPO00619–0623(CO92) YPO00624–0631(CO92) YPO0632–0642(CO92)

RD12 RD13

YPO0738–0754(CO92) YPO1165–1172(CO92)

Yes Yes

YPO0744 YPO1167

RD14 RD15

YPO1286–1289(CO92) YPO1902–1967(CO92)

No Yes

YPO1287 YPO1908 and YPO1954

RD16 RD17 RD18 RD19 RD20 RD21 RD22

YPO2181(CO92) YPO2267(CO92) YPO2271–2281(CO92) YPO2315(CO92) YPO2373–2374(CO92) YPO2375–2376(CO92) YPO3254–3279(CO92)

No Yes Yes Yes No Yes No

YPO2181 YPO2267 YPO2273 YPO2315 YPO2373 YPO2375 YPO3257 and YPO3277

RD23 RD24

YPO3553(CO92) YPO3607–3612(CO92)

Flagellins Dehydrogenase, regulatory proteins, transport protein, regulatory protein, membrane protein, xanthosine utilization Oxidoreductase, dehydrogenase family protein The pgm locus. High-pathogenicity island (HPI), fimbriae gene cluster, two component regulatory system and haemin storage (hms) Membrane protein Pseudogene Prophage Exported protein Lipoprotein and regulatory protein Aldo/keto reductase Amino acid utilization system, autotansporters, multidrug resistance proteins, ATPase, transferase, lipoprotein, membrane protein and regulatory proteins Enhancing lycopene biosynthesis protein Exported protein, regulatory protein and hypothetical proteins

No No

YPO3553 YPO3607

The genes are named with CO92 or 91001 gene definition. RD04 contains the genes selected from plasmid pMT1 (CO92) except those included in RD05 and RD06. Genes included in RD07, RD08 and RD09 are 91001-unique pMT1 genes compared with CO92. RD10 is a 91001-unique chromosomal gene cluster compared with CO92. Plasmid pCRY (RD01) is a novel cryptic plasmid identified in Y. pestis 91001. This plasmid is absent in all the 19 vaccine strains, while of the thirty six natural isolates studied, only two harbor this plasmid.

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hexa-ratios of each gene were averaged. Spots displaying low hybridization signals—the lowest 10% based on Cy3 normalized medians—were filtered out; spots with bad data because of slide abnormalities were also discarded. The efficacy of the DNA microarrays was further assessed by the hybridization results of ‘82009 DNA versus Reference DNA’, ‘91001 DNA versus Reference DNA’ and ‘Reference DNA versus Reference DNA’. Ninety-nine percent of the spots gave correct predictions of the presence or absence of the corresponding genes. The remaining 1% spots gave false predictions and were rejected from the analysis. In the end, 3661 genes were included in the datasets. Log values lower than −1 were taken as defining the absence of a gene in the vaccine strains. This cutoff value was empirically derived from the hybridizations of ‘82009 DNA versus Reference DNA’, ‘91001 DNA versus Reference DNA’ and ‘Reference DNA versus Reference DNA’. Log values larger than 0.75 were taken as identifying the genes amplifications based on the microarray data of RD10 (see below) seen in strain 91001, 82009, Reference (91001 + 82009) and all the vaccine strains. RD10 is a 91001-unique chromosomal gene cluster compared with 82009. Thus, the DNA quantity of RD10 in 91001 is twice as many as those in Reference DNA, which is a model paradigm of genes amplification.

lected genes from all the live vaccines analyzed. Furthermore, these representative genes were used to screen for the distribution of these RDs in thirty six natural isolates of Y. pestis by PCR methods. The 25 ␮l of PCR reaction mixture contained 50 mmol/l KCl, 10 mmol/l Tris–HCl (pH 8.0), 2.5 mmol/l MgCl2 , 0.001% gelatin, 0.1% BSA, 100 ␮mol/l of each dATP, dCTP, dGTP and dTTP, 0.3 ␮mol/l of each primers, 1 U of Taq DNA polymerase (MBI), and 10 ng of template DNA. The amplification was carried out in a DNA thermocycler (Biometra UNOII) with a predenature at 95 ◦ C for 3 min, followed by 30 cycles of 94 ◦ C for 30 s, 60 ◦ C for 30 s and 72 ◦ C for 1 min. A final 5 min elongation at 72 ◦ C was performed after the last cycle to ensure the complete extension of the amplicons. After amplification, 10 ␮l of each PCR product was observed via 1.5% agarose gel electrophoresis with ethidium bromide staining.

3. Results and discussion 3.1. Gene loss in plague live vaccines Twenty four genomic regions consisting of contiguous genes (four of them contain only one gene) were identified by microarray analysis to be absent in one or more vaccine strains (see Table 2). Each of these regions was referred to as a “RD,” a term used in a study of genome variations between M. tuberculosis, M. bovis and BCG [8]. Each of these RDs was further validated by PCR methods. Figs. 1 and 2 show the distribution of these RDs in the 19 vaccine strains tested.

2.4. PCR validation and screening

RD01 RD02 RD03 RD04 RD05 RD06 RD07 RD08 RD09 RD10 RD11-1 RD11-2 RD11-3 RD11-4 RD12 RD13 RD14 RD15 RD16 RD17 RD18 RD19 RD20 RD21 RD22 RD23 RD24

To validate the microarray results about the RDs, one or more genes were chosen from each RD to represent the relevant whole-RD (see Table 2). Gene-specific primers utilized in micorarray construction were reused to amplify these se-

EV76 EV76 (B.SHU) EV EV (SHU) EV (Yuan) EV76 (Paris) EV40 Tjiwidej (Rough) Tjiwidej (Smooth) M23 A1122 P175 Sussia M.II Otten 55 105 410041 P2

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Fig. 1. Presence or absence of RDs in the 19 live vaccines studied. Black filled rectangle denotes the absence of an RD and an empty space the presence. The three grey filled rectangles are somewhat of complexity; for these three RDs the microarray data indicate their absence in the relevant vaccine strains (see Fig. 2), but PCR validation data suggest their presence.

D. Zhou et al. / Vaccine 22 (2004) 3367–3374

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these deletion events have already taken place in the progenitors.

1 0.75 0.5

Log(2.5) ratio

0.25 0 -0.25 -0.5

CO 9

-0.75 -1 -1.25 -1.5

CO 9 2 -YP

2-Y

PP CP 1

. 09

c

P CP 1 .03

-1.75

Gene definition 1 0.75 0.5 0.25 0 -0.25 -0.5 -0.75 -1 -1.25 -1.5

86

CO

27 9 -YP O3

1 0.75 0.5 0.25 0 -0.25 -0.5 -0.75 -1 -1.25 -1.5 -1.75

CO 92 -Y PO 32 54

Log(2.5) ratio

9 PO1 28 CO 92 -Y

Gene definition

(b)

(c)

12 O YP 92

CO 9 2

Log(2.5) ratio

(a)

Gene definition

Fig. 2. (a–c) Graphic representation of microarray data of RD02 (YPPCP1.03–1.09c) in strain Tjiwidej (smooth), RD14 (YPO1286–1289) in strain Otten, and RD22 (YPO3254–3279) in strain EV. The genes are organized in x-coordinate with respect to the CO92 genome order, whereas the log (2.5) ratio of intensity (Test DNA normalized intensity/Reference DNA normalized intensity) in y-coordinate. The microarray data of RD02, RD14 and RD22 indicate their absence in the relevant vaccine strains, but PCR validation data suggest their presence. The rational explanation is that the bacterial culture analyzed contains diversified clones that undergone different genetic mutations, i.e., a majority of microbes in the culture have undergone the deletion of the relevant genomic regions (RD) while the remaining ones have not. In a previous study, the intra-genomic rearrangements were identified in similar way [13]. The authors designed PCR primers to test for a predicted inversion in Y. pestis CO92. It was discovered that the two orientations of the inversion are present simultaneously in the same DNA preparation. These results demonstrate that the Y. pestis genome is fluidic, and capable of frequent intra-genomic recombination and deletion in vitro.

3.1.1. Chromosomal RDs shared by vaccine strains and natural isolates Beside the pgm locus (RD15), there were additional fourteen RDs identified in chromosome. Seven of them (RD10, RD12, RD13, RD17, RD18, RD19 and RD21) are shared by the vaccine strains and the natural isolates analyzed in this work. Live vaccine strains were derived from the fully virulent progenitors by serial in vitro passage and storage. The seven chromosomal RDs shared by the vaccine strains and the natural isolates are in all probability not the outcome of the in vitro processes, i.e.,

3.1.2. Chromosomal RDs unique to vaccine strains Another six chromosomal RDs (RD14, RD16, RD20, RD22, RD23 and RD24) are unique to the vaccine strains. These RDs are likely to be lost during the in vitro passage or storage processes. Live vaccines are generally cultivated in artificial media. The plenteous nutrients and the gentle microenvironment artificially supplied will alleviate greatly the metabolic burdens that the bacteria will encounter in the host. According to the microbial minimalism theory [15,16], genes nonessential for bacterial survival have their bias to be lost. We discovered not a massive, as expected, but a few deletion events in chromosome in the live plague vaccines studied, although most of these strains have lived a ‘well-off’ life in vitro for several decades. Deletions detected in chromosome reflect a reductive adaptation of the live plague vaccines to laboratory conditions. The remaining RD11, YPO0568–0642 in CO92 genome order, is a special one because a partial region of this RD was found to be absent in the natural isolates. This region was further identified as YPO0621–0636 by probing the microarrays with the genomic DNA from a natural isolate (data not shown). The RD11 can be assigned into four parts (RD11-1, RD11-2, RD11-3 and RD11-4) for their divergent distribution in the 19 vaccine strains studied (see Fig. 1). These results indicate this island is variable in genome content within not only vaccine strains but also natural isolates. The RD11 is a virulence-related island that encodes adhesion, autotransporter, protein kinase, resistance proteins and secreted proteins [13]. The absolute or partial loss of the RD11 may affect the virulence and/or immunogenicity of the relevant vaccine strains. 3.1.3. Plasmids and pgm locus Y. pestis normally carries three prototypical virulence plasmids: a 9.5 kb plasmid pPCP1, a 110 kb pMT1 and a 70 kb pCD1 [17]. It is well-known that Y. pestis plasmids are highly unstable under in vitro conditions. Eleven of the vaccine strains studied lost one or more of the three whole-plasmids (see Fig. 1). Plasmids pMT1 is the most divergent one among the three plasmids. It can be assigned into six RDs (RD04 to RD09). Loss of one or more of these RDs occurred in each of the vaccine strains tested. A live plague vaccine must possess a spectrum of protective antigens (e.g. F1 antigen and V antigen) to provide a potent immunogenicity and the broad and long-term protection. Structural genes for F1 and V are located in plasmid pMT1 and pCD1, respectively. Both F1 and V, playing key roles in Y. pestis virulence, are antigenic proteins on cell surface [18–21]. Recombinant F1 and V proteins both induce host immune responses which consistently provide protection against challenge with Y. pestis [4,5]. Plague vaccine strains must contain the F1 antigen and the V antigen because they are thought to play indispensable roles in vaccine

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Table 3 Amplified genomic regions in the live vaccines studied Strain

Genomic region

EV76 EV EV (SHU) EV (Yuan) M23 Otten

YPO4117–4130; YPO0001–0168 YPO0032–0093; YPO3540–3589 YPO0033–0090 Plasmid pPCP1 YP4106–4130; YPO0001–0090, YPO2271–2281 YPO0691–0695; YPO1005–1007

immunogenicity [3,4,12]. Eleven of the 19 vaccine strains in this study lost F1 operon and/or lcrV gene (see Fig. 1). The value of these vaccine strains is seriously questioned. The pgm locus is a 102 kb unstable DNA region, embedded between two IS100 elements in the same orientation, in chromosome [22,23]. The recombination between the two IS elements will delete this locus [24]. The pgm locus is an established virulence-related gene cluster consisting of several distinct parts: the high-pathogenicity island (HPI), a fimbriae gene cluster, a BvgAS-like two component regulatory system and the haemin storage (hms) locus [22,23]. The pgm− strains are attenuated in mammals injected s.c. and the reduced virulence is attributed primarily to the loss of the iron storage and uptake system encoded by HPI [22,25]. It was thought that a live plague vaccine must be pgm− [26]. All the vaccine strains analyzed in this study have undergone the loss of the pgm locus (see Fig. 1). Although the reported safety of the pgm− strains given i.p., s.c., and i.v. [3], Welkos et al. described the pgm− derivative of a full-virulent strain CO92 was clearly virulent for monkeys by the aerosol route [27]. The authors further discovered the pgm− pla− mutant appeared to be more attenuated than either the pgm− mutant or the pla− mutant. Pla is a surface protein with proteolytic, adhesive, and invasive functions that are important for Y. pestis to disseminate from peripheral infection routes and cause systemic infections [28–30]. Recombinant Pla protein is able to induce the host immune response [31], but the reports on its protective efficacy are still not found. It seems that a pgm− pla− F1+ V+ strain has potential as a safer vaccine [27], but this kind of live vaccine is not found in this study. 3.2. Gene amplification in vaccine strains Several large regions were identified to be amplified in the genomes according to the microarray data (see Table 3). Amplification of certain genes can occur when a gene is under strong selection [32]. Almost all the amplified chromosomal regions are bordered by IS elements on one or both ends. The duplication events resulted from IS mediated recombination may lead to the generation of these amplifications. The region of YPO0691–0695 is a portion of a virulence-related island identified in CO92 [13]. While YPO1005–1007 contains three genes encoding the putative antigenic leucine-rich repeat proteins. The amplification of

the plasmid pPCP1 in strain EV (Yuan) may be due to the generation of a dimmer of this plasmid [33]. The genomic variability of these antigen- or virulence-related genes may influence the immunogenicity and/or pathogenicity of the relevant vaccine strains. 3.3. Genomic variability within the vaccine derivants Girard and Robic [11] isolated a strain, named EV, from a human case of bubonic plague in Madagascar in 1926. After 6 years of in vitro passages they obtained an attenuated strain EV76. When used in human immunization in Madagascar, the EV76 vaccine could provide protection against both bubonic and pneumonic plague for human. After then, many derivants of EV vaccines were produced and preserved in the former USSR, the former French colonies, China, Vietnam, and United States [25,34–37]. In this study, seven EV derivants were analyzed. Seven deletions and five amplifications were divergently found in these strains. The genomic differences in the EV vaccines will certainly affect their phenotypes including the immunogenic potency. For example, the EV40 vaccine has lost plasmid pPCP1 and pCD1, and the F1 and Ymt operon on plasmid pMT1 simultaneously. Apparently, this strain is absolutely worthless. Hinchliffe et al. hybridized an EV76 strain with a CO92 gene-specific DNA microarray [38]. Beside the deletion of YPO1165–1172 shared by our results, they still identified a deletion of genomic region containing only one gene YPO0599. YPO0599 encodes a putative adhesin that could not be enriched by passage through mice in Y. pestis [38]. PCR amplifications were performed to screen for the distribution of this gene in the vaccine strains used in our study and no strain was found to lose this gene. In 1936, Otten developed and introduced his two live vaccine strains: the Tjiwidej (smooth) and the Tjiwidej (rough) [12]. The two strains could be easily differentiated from their colony shapes in agar-plate cultures: the former is smooth and the later rough. Our microarray and PCR data revealed that the Tjiwidej (rough) has lost the F1 operon that accounts for the roughness of its colony appearance, for the loss of its ability to assemble F1 capsular on the cell surface. All of the EV strains tested in this study except EV40 harbor both the F1 operon and the lcrV gene. Both of the Tjiwidej strains lost the lcrV gene. The F1 operon and several additional RDs was found to be further lost in the Tjiwidej (rough) (see Fig. 1). These genomic characteristics may account for the following facts: the EV strains are the most immunogenic live vaccines used by far; the protective effectiveness of the EV strains was superior to that of the Tjiwidej (smooth) when used in human immunization in China; the Tjiwidej (smooth) was superior to the Tjiwidej (rough) when used in animal immunization [12]. Both of the Tjiwidej strains should never be used on human subjects for they are V/F1 incomplete, although it was described by Otten [12] that the Tjiwidej strains had the high efficacy in reducing human plague morbidity on the island of Java.

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The different deletion and amplification profiles will account for the variability of the immunogenic and protective efficacy of these live vaccines. Highly immunogenic plague vaccines often cause unpleasant local and systemic reactions with varying intensity and severity, resulting in an overall morbidity greater than that observed with other bacterial vaccines. The immunogenic proteins of Y. pestis including the protective antigens of F1 and V are usually the virulence factors located on or exported out of the bacterial cell surface [39,40]. The adverse effects of live vaccine, together with the long-term questioned safety issue, are apparently related to these virulence-related antigens but providing protective potency. Loss of the genomic regions especially those harboring virulence-related genes may reduce the pathogenic risk, but it will depress the bacterial ability to survival within the hosts, and will lead to the vaccine strains absolutely worthless if the lost genes encode protective proteins. The requirements to minimize adverse effects and to maintain protective potency seems to be two incompatible aspects of one thing.

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